It has, for some time been recognized that while the production of a desired amino acid sequence in procaryotic hosts can be effected by transforming procaryotes with expression vectors containing the appropriate coding sequences under control of procaryotic-compatible promoters, and, desirably, terminators, the resulting proteins may lack the three-dimensional configuration of corresponding proteins produced in their native hosts. Typically, mammalian or other heterologous proteins produced by procaryotic hosts are synthesized in relatively insoluble form, often in the form of particles visible under microscopic examination, said particles variously referred to as "inclusion" or "refractile" bodies.
These insoluble proteins may be successfully identified by immunoprecipitation with antibodies raised against the native forms of the protein but may nevertheless be deficient in their biological activity, presumably due to incorrect folding (see, for example, EPO Publication 114,506, published 8 Jan. 1984), It has been assumed that some of these problems could be eliminated by causing the protein to be secreted either into the periplasm, in the case of gram-negative organisms, or into the medium in the case of gram-positives, presumably because transport through the cellular membrane would result in the proper folding. Indeed, in some instances it has been found possible to prepare soluble, biologically active recombinant proteins using this approach.
Particular instances in which standard techniques of bacterial expression for heterologous proteins have resulted in products which are relatively insoluble and not properly biologically active include standard methods for producing interleukin-2 (IL-2), .beta.-interferon (IFN-.beta.), and ricin A. In the case of ricin A, material obtained by using expression vectors which are plasmids placing the ricin A coding sequence under the control of the trp or P.sub.L promoter, standard "work horse" promoters used in bacterial expression, results in production of a ricin A product which cannot readily be solubilized in the absence of detergents, and which, therefore, does not exhibit cytotoxic activity when conjugated with antibodies to obtain immunoconjugates. Indeed, such immunoconjugates are not cytotoxic even in vitro. The ability of the recombinant ricin A to immunoprecipitate with antibodies raised against the native material is retained, as is the enzymatic ability of the ricin A to inhibit protein synthesis in the standard rabbit reticulocyte assay. It is, therefore, concluded that methods for expression of ricin A using these approaches results in a product which requires additional manipulations to configure the molecule in a three-dimensional array that will enable it to function as a portion of an immunotoxin. Purification procedures cannot readily be applied to the material which has been solubilized by the aid of detergent, and, indeed, it has not proved possible to remove the detergent from the solubilizing solution and retain this material in solution.
By using the expression system of the invention, however, the recombinantly produced ricin A remains soluble in the sonicate from whole cells, and can readily be purified in the absence of detergent. Immunoconjugates prepared with the thus-purified ricin A are cytotoxic both in vivo and in vitro.
An attempt was made to utilize the alkaline phosphatase (phoA) leader and N-terminal sequence to effect secretion of a foreign polypeptide by Ohsuye, K., et al, Nucleic Acids Res (1983) 11:1283. The coding sequence for .alpha.-neoendorphin, a decapeptide hormone, was synthesized in vitro and ligated into vectors so as to produce fusion proteins of the endorphin with the ma)or portion of the alkaline phosphatase N-terminal sequence and leader. The resulting chimeric proteins were processed, but not transported into the periplasm.
It has now been found that the DNA sequence encoding certain bacterial leaders, notably the alkaline phosphatase-encoding leader, are capable of conferring solubility and biological activity characteristics on desired heterologous proteins independently of effecting passage through the membrane. This capacity of the bacterial leader sequence-encoding DNA may be utilized to provide directly the desired forms of proteins previously produced only in more intractable conformations.